Glucose and other reducing sugars react and bind covalently to proteins, lipoproteins and DNA by a process known as non-enzymatic glycation. Glucose latches onto tissue proteins by coupling its carbonyl group to a side-chain amino group such as that found on lysine. Over time, these adducts form structures called advanced glycation endproducts (AGEs) (protein-aging). These cross-linked proteins stiffen connective tissue and lead to tissue damage in the kidney, retina, vascular wall and nerves. The formation of AGEs on long-lived connective tissue accounts for the increase in collagen cross-linking that accompanies normal aging which occurs at an accelerated rate in diabetes.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of a variety of debilitating diseases such as diabetes, atherosclerosis, Alzheimer""s and rheumatoid arthritis, as well as in the normal aging process. Most recent researchers confirm a significant role of the accumulation of AGE cross-links in promoting the decreased cardiovascular compliance of aging (Asif et al., 2000; Vaitkevicius et al., 2001). The process of AGE formation on arterial wall matrix proteins may be related to the development of atherosclerosis in many different ways, such as generation of free radicals (ROS) during the glycation process, inhibition of a normal network formation in collagen by AGE accumulation (Brownlee, 1994), and increased adhesion of monocytes (Gilcrease and Hoover, 1992).
The hallmark Diabetes Control and Complications Trial (DCCT) demonstrated that normalization of blood glucose control by intensive insulin therapy reduces the risk of development of diabetic complications (Diabetes Control and Complications Trial Research Group, 1993). However, intensive insulin therapy neither prevents nor cures complications. Thus, a large number of patients still are prone to develop vascular complications, and additional pharmacological approaches to prevent these complications are desirable.
More recently, several promising therapeutic drugs that could inhibit or break the AGE crosslinks in tissues and cells, and thus prevent these complications, have been reported. Both inhibitors of AGE formation and AGE-breakers not only may have a beneficial effect in reducing these complications, AGE-breakers may cure the disease by removing AGEs from damaged tissues and cells.
Aminoguanidine is a prototype of xe2x80x9cglycation inhibitorsxe2x80x9d. These inhibitors may find therapeutic use in preventing diabetic complications and in delaying normal aging. In addition to aminoguanidine, a large number of much more potent inhibitor compounds have been introduced by us and others recently (Rahbar et al., 1999; Rahbar et al., 2000a; Rahbar et al., 2000b; Kochakian et al., 1996; Khalifah et al., 1999; Soulis et al., 1999; Forbes et al., 2001).
Investigation for selectively cleaving and severing the existing AGE-derived cross-links on tissue proteins by pharmacological strategies has been started more recently. N-phenacylthiazolium bromide (PTB) and ALT 711 have been reported to break AGE cross-links in vitro and in vivo. The introduction of PTB, the first AGE-breaker which was introduced in 1996, generated excitement among the researchers in this field. However, PTB was used at nonphysiological concentrations (10-30 mM), and was observed to degrade rapidly in vitro (Thomalley and Minhas, 1999). Additionally, contrasting results were observed on diabetic rats treated with PTB used at the same concentration of 10 mg/kg daily (Cooper et al., 2000; Oturai et al., 2000). Although the more stable PTB derivative ALT711 has demonstrated AGE-breaking activities both in vitro and in vivo (Vasan et al., 1996; Rahbar et al., 1999), a recent report by Yang et al. (2000) found that ALT711 was not effective in cleaving crosslinks formed in skin and tail collagen of diabetic rats.
The Diabetes Control and Complications Trial (DCCT), has identified hyperglycemia as the main risk-factor for the development of diabetic complications (Diabetes Control and Complications Trial Research Group, 1993). Ever increasing evidence identifies the formation of advanced glycation endproducts (AGEs) as the major pathogenic link between hyperglycemia and the long-term complications of diabetes, namely nephropathy, neuropathy and retinopathy (Makita et al., 1994; Koschinsky et al., 1997; Makita et al., 1993; Bucala et al., 1994; Bailey et al., 1998).
Nonenzymatic glycation is a complex series of reactions between reducing sugars and amino groups of proteins, lipids and DNA, which lead to browning, fluorescence, and cross-linking (Bucala and Cerami, 1992; Bucala et al., 1993; Bucala et al., 1984). The reaction is initiated with the reversible formation of a Schiff""s base, which undergoes a rearrangement to form a stable Amadori product. Both the Schiff""s base and Amadori product further undergo a series of reactions through dicarbonyl intermediates to form advanced glycation endproducts (AGEs).
In human diabetic patients and in animal models of diabetes, these nonenzymatic reactions are accelerated and cause accumulation of glycation products on long-lived structural proteins such as collagen, fibronectin, tubulin, lens crystallin, myelin, laminin and actin, and in addition on several other important biological molecules such as hemoglobin, albumin, LDL-associated lipids and apoprotein. Most recent reports indicate that glycation inactivates metabolic enzymes (Yan and Harding, 1999). The structural and functional integrity of the affected molecules, which often have major roles in cellular functions, become perturbed by these modifications with severe consequences on affected organs such as kidney, eye, nerve, and micro-vascular vessels (Boel et al., 1995; Silbiger et al., 1993; Vlassara et al., 1995; Horie et al., 1997; Matsumoto et al., 1997; Soulis-Liparota et al., 1991; Bucala, 1997; Bucala and Rahbar, 1998; Park et al., 1998). Recent reports indicate glycation to affect metabolic enzymes, high-density lipoproteins and IgG molecules (Yan and Harding, 1999; Lapolla et al., 2000; Lucey et al., 2000; Schalkwijk et al., 1998; Hedrick et al., 2000). The glycation-induced change of immunoglobin G is of particular interest. Recent reports of glycation of Fab fragment of IgG in diabetic patients suggest that immune deficiency observed in these patients may be explained by this phenomenon (Lapolla et al., 2000). Furthermore, an association between IgM response to IgG damaged by glycation and disease activity in rheumatoid arthritis have been reported recently (Lucey et al., 2000). Also, impairment of high-density lipoprotein function by glycation has been reported recently (Hedrick et al., 2000).
Direct evidence indicating the contribution of AGEs in the progression of diabetic complications in different lesions of the kidneys, the rat lens, and in atherosclerosis has been recently reported (Vlassara et al., 1995; Horie et al., 1997; Matsumoto et al., 1997; Soulis-Liparota et al., 1991; Bucala, 1997; Bucala and Rahbar, 1998; Park et al., 1998). Several lines of evidence indicate the increase in reactive carbonyl intermediates (methylglyoxal, glyoxal, 3-deoxyglucosone, malondialdehyde, and hydroxynonenal) is the consequence of hyperglycemia in diabetes. This xe2x80x9ccarbonyl stressxe2x80x9d leads to increased modification of proteins and lipids, followed by xe2x80x9coxidative stressxe2x80x9d and tissue damage (Baynes and Thorpe, 1999; Onorato et al., 1999; McLellan et al., 1994).
Methylglyoxal (MG) has recently received considerable attention as a common mediator to form AGEs. In patients with both insulin-dependent and non-insulin dependent diabetes, the concentration of MG was found to be increased 2-6 fold (Phillips and Thornalley, 1993; Beisswenger et al., 1998). Furthermore, MG has been found not only as the most reactive dicarbonyl AGE-intermediate in cross-linking of proteins, a recent report has found MG to generate reactive oxygen species (ROS) (free radicals) in the course of glycation reactions (Yim et al., 1995).
An intricate relationship between glycation reactions and xe2x80x9coxidative stressxe2x80x9d has been postulated (Baynes and Thorpe, 1999). Nature has devised several humoral and cellular defense mechanisms to protect tissues from deleterious effects of xe2x80x9ccarbonyl stressxe2x80x9d and accumulation of AGEs. These include the glyoxylase system (I and II) and aldose reductase catalyze the deglycation of methylglyoxal (MG), the most common reactive intermediate of AGEs (Phillips and Thornalley, 1993; Beisswenger et al., 1998; Yim et al., 1995), to D-lactate. Additionally, a novel class of enzymes found in Aspergillus, called amadoriases, was found to catalyze the deglycation of Amadori products (Takahashi et al., 1997). Furthermore, several AGE-receptors have been characterized on the surface membranes of monocyte, macrophage, endothelial, mesangial and hepatic cells. One of these receptors, RAGE, a member of the immunoglobulin superfamily, has been found to have a wide distribution in tissues (Schmidt et al., 1994; Yan et al., 1997). MG binds to and irreversibly modifies arginine and lysine residues in proteins. MG modified proteins have been found as ligands for the AGE receptor (Westwood et al., 1997) indicating that MG modified proteins are analogous (Schalkwijk et al., 1998) to those found in AGEs. The discovery of various natural defense mechanisms against glycation and AGE formation suggests an important role of AGEs in the pathogenesis of vascular and peripheral nerve damage in diabetes. Most recently, the effects of MG on LDL have been characterized in vivo and in vitro (Bucala et al., 1993).
Lipid peroxidation of polyunsaturated fatty acids (PUFA), such as arachidonate, also yield carbonyl compounds. Some are identical to those formed from carbohydrates (Al-Abed et al., 1996), such as MG and glyoxal (GO), and others are characteristic of lipid, such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) (Requena et al., 1997). The latter of the carbonyl compounds produce lipoxidation products (Al-Abed et al., 1996; Requena et al., 1997). A recent report emphasizes the importance of lipid-derived MDA in the cross-linking of modified collagen and in diabetes mellitus (Slatter et al., 2000). A number of fluorescent and non-fluorescent AGE compounds that are involved in protein cross-linking have been characterized (Baynes and Thorpe, 1999) (see Table 1). In addition to glucose derived AGE-protein cross-links, AGE cross-linking also occurs between tissue proteins and AGE-containing peptide fragments formed from AGE-protein digestion and turnover. These reactive AGE-peptides, now called glycotoxins, are normally cleared by the kidneys. In diabetic patients, these glycotoxins react with the serum proteins and are a source for widespread tissue damage (Schmidt et al., 1994). However, detailed information on the chemical nature of the cross-link structures remains unknown. The cross-linking structures characterized to date (Table 1), on the basis of chemical and spectroscopic analyses, constitute only a small fraction of the AGE-cross-links which occur in vivo, with the major cross-linking structure(s) still unknown. Recently, a novel acid-labile AGE-structure, N-omega-carboxymethylarginine (CMA), has been identified by enzymatic hydrolysis of collagen, and its concentration was found to be 100 times greater than the concentration of pentosidine (Iijima et al., 2000), and has been assumed to be a major AGE-cross-linking structure (Yan et al., 1997).
Seven compounds have been found which are active in breaking AGE-protein cross-links. These compounds are: 1,4-benzene-bis[4-methyleneaminophenoxyisobutyric acid] (LR 102); 4-[(3,5-dichlorophenylureidophenoxyisobutyryl]-4-aminobenzoic acid (LR99); L-bis-[4-(4-chlorobenzamidophenoxyisobutyryl)cystine] (LR20); 4-(3,5-dichlorophenylureido)phenoxyisobutyryl-1-amidocyclohexane-1-carboxylic acid (LR23); methylene bis [4,4xe2x80x2-(2-chlorophenylureidophenoxyisobutyric acid)] (LR90); 5-aminosalicylic acid (5-ASA) (also referred to herein as SMR-5); and metformin (also referred to herein as SMR-12).
In one aspect of the invention, these AGE-breaking compounds are used to break glycation endproducts or cross-linked proteins in an organism by administering to an organism an effective amount of one or more of the AGE-breakers.
In a second aspect of the invention, the deleterious effects of aging in an organism are reversed by administering an effective amount of an AGE-breaker to the organism.
In a third aspect of the invention, complications resulting from diabetes in an organism are reversed by administration of an effective amount of an AGE-breaker to the organism.
In further aspects of the invention, the progress of disease in a patient, wherein the disease can include rheumatoid arthritis, Alzheimer""s disease, uremia, neurotoxicity, or atherosclerosis, is reversed by administration of an effective amount of an AGE-breaker to the patient.